Many flaviviruses such as Dengue virus, West Nile virus, and Yellow Fever virus cause significant human diseases. However, no clinically approved antiviral therapy is available for treatment of flavivirus infections. Therefore, the development of vaccines and antiviral agents for prevention and treatment of flavivirus infections is a clear public health priority. Previously, we found a general mechanism that the flavivirus NS5 sequentially methylates the guanine N7 on the cap and ribose 2'-OH on the first transcribed nucleotide. We also found that the N7 methyltransferase (MTase) is essential for flavivirus replication, since defects in N7 MTase activity lead to defects in viral replication. We also identified a flavivirus-conserved pocket near the methyl- donor SAM-binding pocket. In addition, we identified a lead MTase inhibitor, and found that inhibitors against the N7 activity of the WNV MTase can inhibit viral growth. These results clearly demonstrate that flavivirus MTase is a novel target for development of antiviral therapy. The goal of this application is to study the molecular inhibition of the essential flavivirus MTase. We will accomplish the overall objective of this application by pursuing three specific aims. Aim 1. Characterization of the flavivirus methyltransferase. We will further characterize the flavivirus- conserved pocket near the co-factor SAM-binding site using mutagenesis and reverse genetic approaches. Aim 2. Structure-based design and development of MTase inhibitors. We will perform structure-based design and synthesis of specific inhibitors against flavivirus MTases. Aim 3. Performance of in-depth structure-activity studies and determination of X-ray structures of selected inhibitors to gain molecular insight. We will evaluate the inhibition ability of synthesized MTase inhibitors using in vitro MTase assay, cell-based replicon inhibition assay, and viral reduction assay. Advanced compounds will be further characterized by in vivo animal studies, in-depth drug resistance studies, and co-crystal structure determination of the MTase-inhibitor complexes. The proposed research is innovative and significant. The knowledge acquired from this study will significantly advance our understanding of flavivirus cap methylation and lead to the development of inhibitors of the flavivirus MTase for antiviral therapy.